Influenza viruses are RNA orthomyxoviruses and consist of three types, A, B and C. Whereas influenza viruses of types B and C are predominantly human pathogens, influenza A viruses infect a wide variety of birds and mammals, including humans, horses, marine mammals, pigs, ferrets, and chickens. In animals, most influenza A viruses cause mild localized infections of the respiratory and intestinal tract. However, highly pathogenic influenza A subtypes, such as H5N1, also exist that cause systemic infections in poultry in which mortality may reach 100%. Several subtypes of influenza A viruses also may cause severe illness in man.
Influenza A viruses can be classified into subtypes based on allelic variations in antigenic regions of two genes that encode surface glycoproteins, namely, hemagglutinin (HA) and neuraminidase (NA), which are required for viral attachment and cellular release. Other major viral proteins include the nucleoprotein, the nucleocapsid structural protein, membrane proteins (M1 and M2), polymerases (PA, PB and PB2) and non-structural proteins (NS1 and NS2). Currently, sixteen subtypes of HA (H1-H16) and nine NA (N1-N9) antigenic variants are known in influenza A virus. Influenza virus subtypes can further be classified by reference to their phylogenetic group. Phylogenetic analysis (Fouchier et al., 2005) has demonstrated a subdivision of HAs that falls into two main groups (Air, 1981): inter alia the H1, H2, H5 and H9 subtypes in phylogenetic group 1 and inter alia the H3, H4 and H7 subtypes in phylogenetic group 2 (FIG. 1).
Only some of the influenza A subtypes (i.e., H1N1, H1N2 and H3N2) circulate among people, but all combinations of the 16 HA and 9 NA subtypes have been identified in avian species Animals infected with influenza A often act as a reservoir for the influenza viruses and certain subtypes have been shown to cross the species barrier to humans, such as the highly pathogenic influenza A strain H5N1.
Influenza infection is one of the most common diseases known to man, causing between three and five million cases of severe illness and between 250,000 and 500,000 deaths every year around the world. Influenza rapidly spreads in seasonal epidemics affecting 5-15% of the population and the burden on health care costs and lost productivity are extensive (World Healthcare Organization (WHO)). Hospitalization and deaths mainly occur in high-risk groups (elderly, chronically ill).
Annual epidemics of influenza occur when the antigenic properties of the viral surface protein HA and NA are altered. The mechanism of altered antigenicity is twofold: antigenic shift, caused by genetic rearrangement between human and animal viruses after double infection of host cells, which can cause a pandemic; and antigenic drift, caused by small changes in the HA and NA proteins on the virus surface, which can cause influenza epidemics. The emergence of variant virus strains by these two mechanisms is the cause of influenza epidemics. Three times in the last century, the influenza A viruses have undergone major genetic changes, mainly in their HA-component, resulting in global pandemics and large tolls in terms of both disease and deaths. The most infamous pandemic was “Spanish Flu,” caused by influenza virus H1N1, which affected large parts of the world population and is thought to have killed at least 40 million people in 1918-1919. More recently, two other influenza A pandemics occurred, in 1957 (“Asian influenza,” caused by influenza virus H2N2) and 1968 (“Hong Kong influenza,” caused by influenza virus H3N2), and caused significant morbidity and mortality globally. In contrast to current seasonal influenza epidemics, these pandemics were associated with severe outcomes also among healthy younger persons.
Current approaches to dealing with annual influenza epidemics include annual vaccination, preferably generating heterotypic cross-protection. However, as indicated above, circulating influenza viruses in humans are subject to permanent antigenic changes, which require annual adaptation of the influenza vaccine formulation to ensure the closest possible match between the influenza vaccine strains and the circulating influenza strains.
Although yearly vaccination with the flu vaccine is the best way to prevent the flu, antiviral drugs, such as oseltamivir (TAMIFLU®), can be effective for prevention and treatment of the flu. However, the number of influenza virus strains showing resistance against such oseltamivir is increasing.
An alternative approach is the development of antibody-based prophylactic or therapeutic means to neutralize various seasonal influenza viruses. The primary target of neutralizing antibodies that protect against influenza virus infection is the globular head (HA1 part) of the viral HA protein, which contains the receptor binding site, but is subject to continuing genetic evolution with amino acid substitutions in antibody-binding sites (antigenic drift). Cross-neutralizing antibodies recognizing the more conserved stem-region of HA of influenza A viruses of phylogenetic group 1 (e.g., H1 and H5) have recently been identified (e.g., WO2008/028946). There has, however, been limited success in identifying antibodies that neutralize one or more influenza A virus subtypes of phylogenetic group 2, such as H3 viruses, and their breadth of neutralization is narrow and their potency low.
Antibodies specifically recognizing H3N2 influenza virus strains have been described. Thus, a human monoclonal antibody, C28, capable of binding to and neutralizing several H3N2 influenza virus strains from the years between 1968 and 1980 has been described by Östberg and Pursch (1983). Wang et al. (2010) have described an anti-HA2 murine antibody neutralizing H3 viruses spanning several decades, but which was shown not to neutralize any non-H3 subtype viruses.
Cross-reactive anti-HA2 murine antibodies recognizing HA of the H3 subtype, as well as of the H4 and H7 subtype, and capable of in vitro reducing influenza virus replication of H3 and H4 influenza viruses have been described by Stropkovská et al. (2009). It was demonstrated that the accessibility of the HA2 epitopes to these antibodies in the native virus was low, and that the antibodies have a higher reactivity with HA after its trypsin cleavage and pH 5 treatment (Vare{hacek over (c)}ková et al., 2003a), which may explain the observation that the in vitro inhibition of virus replication (Vare{hacek over (c)}ková et al., 2003b), as well as in vivo potency of these antibodies was relatively low (Gocník et al., 2007).
In WO2009/115972, a human monoclonal antibody, Fab28, is disclosed that recognizes an epitope on the stem region of HA and displays a neutralizing activity against H1N1, but less neutralizing activity against H3N2.